A critical control point for regulating gene expression in eukaryotic cells is during mRNA transcription by RNA polymerase II (Pol II). Non-coding RNA molecules (ncRNAs) have emerged as a new class of transcriptional regulators in a variety of cellular systems. Mouse B2 RNA and human Alu RNA are two such ncRNAs; they function as repressors of mRNA transcription by binding directly to Pol II in response to heat shock, a widely used model system for studying the cellular stress response. The long term goal of research funded by the parent grant is to understand how mammalian ncRNAs that bind to Pol II control transcription in biologically important and medically relevant experimental systems. The proposed work will complement and extend the goals of the parent grant by identifying and investigating new ncRNA/Pol II complexes in cells. Doing so will test the hypothesis that Pol II contains a high-affinity docking site for ncRNAs, and that diverse ncRNAs bind this site and function as trans-regulators of Pol II transcription in different cellular systems. The proposal hs one Specific Aim: Identify and characterize novel ncRNA/Pol II complexes that form under distinct cellular conditions. New macromolecular complexes containing ncRNAs and Pol II will be found by immunoprecipitating Pol II and sequencing the associated RNAs. Two cellular systems will be used, one focusing on a program of differentiation and the other on cell cycle progression. For selected ncRNAs the percent of each ncRNA bound to Pol II, and how this changes under different conditions and cell types, will be experimentally determined. In addition, the contribution the selected ncRNAs make to the overall programs of differentiation and cell cycle progression in the two cell types will be investigated. These studies will move the fields o transcriptional regulation and functional ncRNAs in new directions. A new class of macromolecular complexes in cells will be defined that have potential to control gene expression through novel mechanisms. Results from the proposed work will provide the platform for future lines of research that will investigate how ncRNAs that complex with Pol II control transcriptional activity during programs of cellular differentiation and cell cycle progression.